In this application, we propose to employ bacteriorhodopsin, a light-activated proton pump from Halobacterium salinarium, to manipulate the pH gradient in synaptic vesicles. Synaptic vesicle filling with neurotransmitters is highly sensitive to intravesicular pH, which is regulated by an intrinsic vesicular proton pump, vacuolar ATPase (v-ATPase). Recent studies, including work from our group, suggests that inhibition of v-ATPase by small molecule inhibitors (e.g. bafilomycin) results in fast use-dependent rundown of synaptic responses and blockade of neurotransmitter release. In this project, we will exploit this strict pH-dependence of the synaptic vesicle refilling process by using bacteriorhodopsin targeted to synaptic vesicles in neurons to emulate the effect of v-ATPase inhibitors in a light-induced and rapidly reversible fashion without global changes in the membrane excitability. In addition, targeting of bacteriorhodopsin to other secretory organelles such as lysosomes that critically depend on the intravesicular pH for their proper operation can be a powerful tool to investigate their role(s) in neuronal function. We propose to develop this project in three stages: First, we aim to selectively target bacteriorhodopsin in a functional conformation to synaptic vesicles. Second, we will optimize light-induced proton pump activity of the vesicular bacteriorhodopsin in cultured hippocampal neurons. Finally, we will express optimized bacteriorhodopsin constructs in vivo using specific neuronal promoters in Drosophila for light-induced manipulation of Drosophila behavior. Taken together the research proposed here has significant potential in bridging synaptic functional studies in vitro and information processing in the intact brain. This approach will enable acute manipulation of synaptic inputs into a particular area of the brain to test how they may influence function as well as behavioral output. PUBLIC HEALTH RELEVANCE: In this application we propose to target bacteriorhodopsin, a light-activated proton pump from Halobacterium salinarium, specifically to synaptic vesicles by engineering fusion proteins to impair synaptic transmission in a reversible and light-induced fashion. Successful completion of this project will yield important tools that can enable synapse specific manipulation of neuronal circuits.